In higher eukaryotic cells, duplication and expression of genetic information occur within the cell nucleus. At the onset of mitosis, the interphase nucleus disassembles and the duplicated chromatin is packaged into mitotic chromosomes before being transported into two daughter cells. This process, mitotic chromosome condensation, is believed to be an essential process for maintaining the integrity of genetic information during mitosis. Our overall goal is to determine the molecular mechanisms responsible for the dynamic changes of higher-order chromosome structure during the cell cycle. This-information is important for our understanding of the mechanisms of carcinogenesis in which the instability of genetic information, often accompanied with chromosomal anomalies, results in malignant transformation. Using a cell-free system derived from Xenopus (toad) egg extracts, we have recently identified a novel chromosomal protein (termed XCAP-C/E) that appears to be a key player in mitotic chromosome condensation. The specific aim of this proposal is to determine how this protein works in chromosome assembly process, and how the activity is regulated during the cell cycle. First we will purify XCAP-C/E from the egg extracts, and determine how it interacts with DNA. The role of ATP-binding/hydrolysis by XCAP-C/E in its DNA-binding activity will be determined. Such basic information will provide a framework for our understanding of the molecular function of XCAP-C/E. We will then determine the mechanisms of cell cycle regulation of XCAP-C/E by examining both cell cycle-specific modification and interacting proteins. We will also characterize two polypeptides specifically associated with XCAP-C/E and determine their functional roles. These experiments will allow us to understand how dynamic organization of chromosomes is modulated during the cells cycle. Finally, we will use purified XCAP-C/E and the nucleosome, a basic unit of eukaryotic chromatin, to set up a model system for reconstitution of higher-order chromosome structures in vitro.